Gli autori avrebbero riconosciuto Scott Howard e Abigail Marinack per il sostegno generale e il coordinamento di questo lavoro.

Tutti gli studi che hanno coinvolto campioni di urina umana sono stati condotti aderenza alla procedura approvata da Pharmasan / Neuroscience Institutional Review Board (IRB). 1. Determinazione del campione di urina e della determinazione della creatinina (Cr) <ol><li> Raccogli 5 ml dei campioni di urina del mattino successivo dopo ca. 10 ore di digiuno in un tubo di trasporto da 5 mL contenente 250 μL di 3 N HCl come conservante e conservare a -20 ° C fino all&#39;uso. </li><li> Sciogliere e vorticare 5 ml di urina di trasporto Un tubo e centrifugare in una centrifuga ( ad es. Sorvall) (2297 xg, 10 min). </li><li> Aliquota 1 ml di urina due volte dal tubo di trasporto 5 mL. </li><li> Determinare Cr con un metodo della creatinina urinaria 26 . </li></ol> 2. Preparazione dei campioni Standard, IS e Quality Control (QC) <ol><li> Preparare una soluzione di stock di 3 NT a 100 μg / mL in acqua con una fase mobile A (MA) di 0,01% HCOOH e conservare a -20 ° C. </li><li> RendereUna soluzione di lavoro standard di 3-NT a concentrazione di 100 ng / mL diluendo la soluzione di stock di 3-NT di 100 μg / mL con MA. </li><li> Generare standard che vanno da 5 a 2500 pg / mL per diluizione dello standard di lavoro di 100 ng / ml con 0,15 M HCl acidato vuoto in bianco senza 3 NT con campioni in bianco e doppio vuoto. </li><li> Preparare una soluzione a base di IS 13 C 9 -3-NT a 100 μg / mL in acqua con MA e conservare a -20 ° C. </li><li> Realizzare una soluzione di lavoro IS a 500 pg / mL diluendo la soluzione di stock di 100 μg / mL 13 C 9 -3-NT IS con MA. </li><li> Stabilire cinque livelli di campioni di QC che coprono i livelli LLOQ, bassi, medi e alti ( vale a dire , 10, 25, 100, 500 e 1.250 pg / mL), diluendo lo standard di lavoro 3-NT nell&#39;urina vuota acidificata. </li></ol> 3. Procedura di estrazione a fase solida <ol><li> Campioni di urina e di vortice, standard e campioni di QC. </li><li> Aggiungere campioni di urina, standard e QC sampLes (250 μL ciascuno) a 32 pozzetti di una piastra di raccolta a 96 pozzetti puliti da 2 ml. </li><li> Introdurre la soluzione di lavoro di 500 pg / ml IS (50 μL) ad ogni pozzetto, tranne il doppio campione di campioni vuoti. Aggiungere il 0,01% HCOOH (50 μL) al pozzetto campione doppio vuoto. </li><li> Aggiungere acqua LC-MS / MS con 0,1% HCOOH (250 μL). </li><li> Mescolare la miscela sopra con una pipetta a 8 canali tre volte. </li><li> Coprire la piastra fino al caricamento SPE. </li><li> Posizionare una piastra di estrazione MCX da 96 pozzetti e un serbatoio di raccolta su un processore a pressione positiva. </li><li> Condizionare la piastra di estrazione con MeOH fluido (200 μL) attraverso il sorbente. </li><li> Equilibrare con acqua corrente con 2% HCOOH (200 μL) attraverso il sorbente. </li><li> Caricare l&#39;intero volume di ciascuno dei campioni prelevati sulla piastra di estrazione pre-condizionata con una pipetta a 8 canali. </li><li> Impostare una bassa pressione positiva ( ad esempio , 3 psi) sul processore di pressione positivo per consentire la miscelazione a fluireSe il sorbente è lento, regolare la pressione se necessario. </li><li> Lavare i pozzetti con acqua fluente con 2% HCOOH (200 μL) attraverso il sorbente. </li><li> Lavare i pozzetti facendo scorrere il metanolo (200 μL) attraverso il sorbente. </li><li> Lavare i pozzetti con acqua corrente (200 μL) attraverso il sorbente. </li><li> Asciugare completamente i pozzetti con un&#39;alta pressione positiva ( es . 40 psi) sul processore di pressione positiva. </li><li> Sostituire il serbatoio con una piastra di raccolta a 96 pozzetti da 2 ml. </li><li> Applicare 25 mM NH 4 OAc a pH 9 (50 μL) per eluire l&#39;analita conservata e IS dalla piastra di estrazione. </li><li> Pipettare l&#39;acqua LC-MS con 5% HCOOH (50 μL) per neutralizzare l&#39;eluato. </li><li> Mescolare con la pipetta a 8 canali tre volte e inviare alla stazione LC-MS / MS per l&#39;analisi. </li></ol> 4. Analisi LC-MS / MS <ol><li> Misurare con precisione 2,000 ml di acqua LC-MS con un cilindro graduato e trasferirlo in una bottiglia da 2 L. </li><li> PImmergere 200 μL di HCOOH puro nella bottiglia sopra riportata contenente acqua LC-MS. </li><li> Mescolare accuratamente ed etichettare come fase mobile A (MA). Includi le iniziali, la data di preparazione e la data di scadenza. </li><li> Prendere una bottiglia da 2 L di metanolo LC-MS e l&#39;etichetta come fase mobile B (MB) con data di inizio e data di scadenza. </li><li> Impostare la temperatura del campionatore automatico a 4 ° C. </li><li> Posizionare la lastra di raccolta con campioni preparati nell&#39;autocampionatore. </li><li> Posizionare una colonna PFP (150 mm x 2,1 mm id, 3 μm) e una colonna di protezione nel forno. </li><li> Impostare la temperatura del forno a 30 ° C. </li><li> Equilibrare 10 min utilizzando il metodo di acquisizione con l&#39;eluizione del gradiente LC come mostrato nella Tabella 1 . </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Tempo (min) </td><td> Modulo </td><td> eventi </td><td> Parametro </td></tr><tr><td> 0 </td><td> pompe </td><td> Pompa B Conc. </td><td> 5 </td></tr><tr><td> 0.5 </td><td> pompe </td><td> Pompa B Conc. </td><td> 20 </td></tr><tr><td> 1 </td><td> pompe </td><td> Pompa B Conc. </td><td> 50 </td></tr><tr><td> 3 </td><td> pompe </td><td> Pompa B Conc. </td><td> 80 </td></tr><tr><td> 4 </td><td> pompe </td><td> Pompa B Conc. </td><td> 90 </td></tr><tr><td> 4.01 </td><td> pompe </td><td> Pompa B Conc. </td><td> 95 </td></tr><tr><td> 5.5 </td><td> pompe </td><td> Pompa B Conc. </td><td> 95 </td></tr><tr><td> 5.6 </td><td> pompe </td><td> Pompa B Conc. </td><td> 5 </td></tr><tr><td> 7 </td><td> controllore </td><td> Stop </td><td></td></tr></tbody></table> Tabella 1: Condizioni di eluizione del gradiente di cromatografia liquida <ol start="10"><li> Creare un elenco di batch inclusoStandard, QC e campioni di urina. </li><li> Avviare il lotto mediante l&#39;iniezione dei campioni preparati (12 μL). </li></ol> 5. Identificazione del picco, integrazione e processo dei dati <ol><li> Controlla l&#39;acquisizione e l&#39;elaborazione dei dati utilizzando il software. </li><li> Identificare e integrare picchi 3-NT e IS per tutti i campioni. </li><li> Stabilire una curva standard con l&#39;intervallo di 10-2.500 pg / mL per la quantitativa 3-NT mediante regressione lineare del rapporto di area di picco di 3-NT e IS rispetto alla concentrazione nominale 3-NT con un fattore di ponderazione di 1 / x. </li><li> Quantifica tutti i campioni utilizzando la curva standard. </li><li> Determinare se i campioni di QC cadono nell&#39;intervallo stabilito. </li><li> Convertire le concentrazioni rilevate di campioni di urina ai risultati finali nell&#39;unità di nM o nmol / mmol Cr. </li></ol>

<ol>
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Gli autori dichiarano di non avere alcun conflitto di interessi.

Le variazioni sostanziali delle concentrazioni precedentemente riportate in letteratura per i campioni di urina umana libera 3-NT endogene rivelano problemi metodologici associati ai dosaggi disponibili 8 , 9 , 10 , 11 . La precisa determinazione del basso livello basale di 3-NT nelle urine umane rimane un compito impegnativo che richiede particolari precauzioni per la preparazione del campion...

Gli effetti dello stress ossidativo sulla presentazione clinica sono stati spinti in prima linea negli ultimi anni 1 . Uno dei biomarcatori esplorati è la 3-nitrotirosina (3-NT), un prodotto stabile stabile formato quando le specie di azoto reattivo (RNS) interagiscono con la tirosina, un precursore di neurotrasmettitore di catecolamine. Mentre 3-NT può avere un valore clinico come biomarker per RNS in vivo, i cambiamenti sostanziali delle proprietà e delle funzioni della tirosina possono influenzare negativamente le corrispondenti proteine ​​e le funzioni cellulari 1 , 2 . La ricerca emergente ha suggerito che il 3-NT può svolgere un ruolo importante nelle condizioni infiammatorie 3 , nei disturbi neurodegenerativi 4 , 5 , nella malattia cardiovascolare 6 e nel diabete 7 , nonché nelle condizioni correlate allo stress ossidativo. Tuttavia, questi obseLe risposte si basano sui risultati di metodologie prive di sensibilità e / o selettività 8 , 9 , 10 , 11 . Le enormi intervalli di concentrazione di 3 NT per i campioni biologici precedentemente riportati in letteratura rivelano che sono stati associati seri problemi analitici con questi dosaggi e sono necessari miglioramenti tecnici per quantificare accuratamente i livelli di 3 NT e verificarne il ruolo nella patologia di queste condizioni . La quantitazione di 3 NT gratuite in matrici biologiche rappresenta una sfida speciale per l&#39;uomo e lo strumento 8 , 9 , 10 , 11 . In primo luogo, il livello di traccia di 3-NT endogeno richiede una rilevazione ultra-sensibile; In secondo luogo, l&#39;esistenza di numerosi analoghi strutturalmente simili, in particolare la tirosina, che è presenteVasto eccesso, richiede un elevato grado di selettività; In terzo luogo, la formazione artificiale di 3 NT per nitrazione di tirosina con nitrati e nitriti onnipresenti richiede particolare attenzione durante la preparazione del campione per evitare falsi sovrastimazioni di 3 NT. Tra un&#39;ampia varietà di metodologie utilizzate per misurare 3-NT, MS / MS è stato considerato il metodo del gold standard dovuto alla sua maggiore sensibilità e selettività 11 , 12 , 13 , 14 . La cromatografia a gas (GC) accoppiata MS / MS offre la migliore sensibilità, tuttavia i passi indispensabili di derivatizzazione del campione sono troppo noiosi e richiedono molto tempo per essere efficienti per l&#39;utilità clinica 15 , 16 . LC-MS / MS non richiede una derivatizzazione complessa del campione, rendendola l&#39;opzione più promettente. Tuttavia, ci sono diversi ostacoli per superare come il seLa nsitività dei metodi LC-MS / MS riportati in letteratura deve essere migliorata per la misurazione della bassa abbondanza 3-NT 7 , 17 , 18 e il tempo di rotazione relativamente lungo deve essere abbreviato per applicazioni ad alta velocità 12 , 13 , 17 , 19 . Inoltre, quando si considerano le applicazioni cliniche, la matrice biologica utilizzata svolge un ruolo significativo. Deve essere facile e poco costoso ottenere e non invasivo se possibile 20 , 21 , 22 . Il plasma, il campione tradizionalmente usato in letteratura, non è una matrice clinicamente desiderabile, quindi è stata ricercata una metodologia che utilizza urina non invasiva e conveniente. Diversi tentativi di sviluppare relSono state effettuate metodologie LC-MS / MS specifiche e usando l&#39;urina 9 , 10 , 11 . Tuttavia, essi non sono riusciti a essere selettivi, affidabili o efficienti per l&#39;uso clinico. L&#39;efficacia del SPE predominante che utilizza la cartuccia tradizionale a fase inversa (tipo C18) come pulizia del campione per l&#39;analisi 3-NT è stata interrogata e è stato proposto un SPE sequenziale di scambio forte cation (SCX) e C18-OH in fase inversa 6 , 7 , 19 . Un metodo LC-MS / MS, recentemente sviluppato, ha utilizzato un processo di purificazione multipla di C18 SPE manuale, cromatografia liquida preparata ad alta pressione (HPLC) e SPE online per l&#39;analisi di 3-NT 23 . Sebbene questo metodo fosse sufficientemente sensibile per scopi clinici, con un LLOQ di 0,041 nM, il processo di pulizia era intenso e noioso e richiedevaRosso 3 ml di urina, limitandone la sua fattibilità ad alta produttività. Un polimero impressa in molecola è stato impiegato come sorbente SPE per migliorare l&#39;efficienza del processo di pulizia 14 , ma il risultato LLOQ (0,7 μg / mL) non era abbastanza basso per gli esemplari clinici. Un altro metodo ha richiesto la cromatografia bidimensionale (2D) LC-MS / MS e l&#39;immunoaffinita per la pulizia del campione per ottenere un limite di rilevazione (LOD) di 0,022 nM 24 . Mentre tutti questi metodi hanno fatto progressi nella valutazione di 3-NT, nessuno ha raggiunto la sensibilità, l&#39;affidabilità e l&#39;efficienza necessaria per le applicazioni cliniche. Per studiare la patologia del 3-NT libero e il suo ruolo di biomarker dello stress ossidativo in ambito clinico, abbiamo sviluppato una metodologia semplice, efficace, precisa e precisa, che consente di applicare applicazioni cliniche ad alto rendimento 25 . Un cation miniaturizzato in modalità mista excHange (MCX) è stata implementata in micropiastre di estrazione a 96 pozzetti per ottenere un semplice ed efficace processo di pulizia e arricchimento di 3 NT in una singola estrazione, superando gli inconvenienti osservati nei metodi esistenti che richiedono derivatizzazione, evaporazione e 2D-LC. La cromatografia liquida con 0,01% HCOOH come additivo in fase mobile ha offerto una risposta di segnale avanzata con un rapido tempo di ciclo. La selettività è stata ulteriormente migliorata attraverso l&#39;applicazione di una soluzione di eluzione NH 4 OAc mite per l&#39;eluizione selettiva di 3-NT e l&#39;uso della transizione MRM per entrambi i 3 NT e lo standard interno (IS). L&#39;effetto della matrice è stato compensato utilizzando una quantità ridotta di una isotopo preferito di 13 C-etero per la quantificazione. Con l&#39;avvento di questa metodologia, i ricercatori e i clinici saranno in grado di verificare il ruolo di 3-NT nelle condizioni cliniche e di esplorare ulteriormente l&#39;impatto dello stress ossidativo.

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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">3-Nitro-L-tyrosine</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">N7389-5g</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">3-Nitro-L-tyrosine-13C9</td>
			<td>Sigma</td>
			<td>652296-5.0mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mass Spec Gold Urine</td>
			<td>Golden West Biologicals</td>
			<td>MSG 5000-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oasis MCX 96-well &#181;Elution plate</td>
			<td style="width:161px;">Waters</td>
			<td>186001830BA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">2mL 96 well collection plate</td>
			<td style="width:161px;">Phenomenex&#160;&#160;</td>
			<td style="width:115px;">AH0-7194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 positive processor</td>
			<td style="width:161px;">Waters&#160;</td>
			<td style="width:115px;">186005521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LC-MS Ultra CHROMASOLV methanol&#160;&#160;</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">14262-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LC-MS Ultra CHROMASOLV water</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">14263-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic acid for mass spectrometry</td>
			<td style="width:161px;">Sigma</td>
			<td>94318-50ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide solution</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">338818-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra PFP propyl columns</td>
			<td style="width:161px;">Restek</td>
			<td style="width:115px;">9179362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5500 Triple quad</td>
			<td style="width:161px;">AB Sciex&#160;</td>
			<td style="width:115px;">/</td>
			<td style="width:263px;">Contact manufacture for more detail</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UFLC-XR</td>
			<td style="width:161px;">Shimadzu&#160;</td>
			<td style="width:115px;">/</td>
			<td style="width:263px;">Contact manufacture for more detail</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Integra 400 Plus&#160;</td>
			<td style="width:161px;">Roche</td>
			<td style="width:115px;">/</td>
			<td style="width:263px;">Urinary Creatinine Jaff&#233; Gen 2 method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCMS certified 12 x 32mm screw neck vial</td>
			<td style="width:161px;">Waters</td>
			<td>600000751CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCGC certified 12 x 32mm screw neck total recovery vial</td>
			<td style="width:161px;">Waters</td>
			<td>186000384C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL transport tube</td>
			<td style="width:161px;">Phenix</td>
			<td>TT-3205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">50 mL Centrifuge tube</td>
			<td style="width:161px;">Crystalgen&#160;</td>
			<td style="width:115px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">15 mL Centrifuge tube</td>
			<td style="width:161px;">Crystalgen&#160;</td>
			<td style="width:115px;">23-2266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eLine electronic pipette</td>
			<td style="width:161px;">Sartorius</td>
			<td style="width:115px;">730391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge centrifuge&#160;</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td style="width:115px;">A46474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OHAUS balance&#160;&#160;</td>
			<td style="width:161px;">Kennedy Scales, inc.</td>
			<td style="width:115px;">735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer&#160;</td>
			<td style="width:161px;">Bernstead Thermolyne</td>
			<td style="width:115px;">M16715</td>
			<td></td>
		</tr>
	</tbody>
</table>

integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

La libera 3-nitrotirosina (3-NT) è stata ampiamente utilizzata come possibile biomarker per lo stress ossidativo. Sono stati riportati livelli aumentati di 3 NT in una grande varietà di condizioni patologiche. Tuttavia, i metodi esistenti non dispongono di sufficiente sensibilità e / o specificità necessarie per misurare in modo affidabile il basso livello endogeno di 3 NT e sono troppo ingombranti per le applicazioni cliniche. Quindi, è urgentemente necessario un miglioramento analitico per quantificare accuratamente i livelli di 3 NT e verificare il ruolo del 3-NT in condizioni patologiche. Questo protocollo presenta lo sviluppo di una nuova rivelazione di spettrometria di massa tandem (LC-MS / MS) cromatografica a liquido combinata con un&#39;estrazione miniaturizzata di fase solida (SPE) per la misurazione rapida ed accurata di 3-NT nelle urine umane come biomarker non invasivo Per lo stress ossidativo. SPE usando una piastra a 96 pozzetti ha semplificato notevolmente il processo combinando l&#39;arricchimento del campione e l&#39;arricchimento dell&#39;analita- zione senza derivatizzazione e fasi di evaporazione, Riducendo il consumo di solventi, lo smaltimento dei rifiuti, il rischio di contaminazione e il tempo complessivo di lavorazione. L&#39;impiego di 25 mM acetato di ammonio (NH 4 OAc) a pH 9 come soluzione di eluizione SPE ha migliorato sostanzialmente la selettività. La risposta al segnale di spettrometria di massa è stata migliorata attraverso la regolazione delle transizioni di monitoraggio delle reazioni multiple (MRM). L&#39;uso di 0,01% HCOOH come additivo su una colonna pentafluorofenile (PFP) (150 mm x 2,1 mm, 3 μm) ha migliorato la risposta del segnale di un&#39;altra 2,5 volte e ha abbreviato il tempo di esecuzione totale a 7 minuti. È stato raggiunto un limite inferiore di quantitativi (LLOQ) di 10 pg / mL (0,044 nM), che rappresenta un significativo miglioramento della sensibilità rispetto ai dosaggi riportati. Questo metodo semplificato, rapido, selettivo e sensibile consente di elaborare due lastre di campioni di urina (n = 192) in un periodo di 24 ore. Considerando le prestazioni analitiche notevolmente migliorate e il campionamento di urina non invasivo e poco costoso, il saggio proposto è vantaggioso per pre-cliniche e clinichestudi.

La figura 1 illustra che 3-NT è completamente cromatograficamente separato da altri analoghi strutturali simili di tirosina sotto la condizione LC ottimizzata, che elimina le interferenze co-eluenti dovute a questi composti eccessivamente eccessivi e quindi aumenta il grado di selettività del dosaggio. Inoltre, l&#39;eluizione di gradiente con 0,01% di HCOOH come additivo in MA e metanolo ad una velocità di flusso di 0,45 ml / min consente un&#39;eluzione...

Watch this Scientific Journal Video about Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications at JoVE.com

Integrazione dell'estrazione miniaturizzata in fase solida e rilevamento LC-MS/MS della 3-nitrotirosina nell'urina umana per applicazioni cliniche | Chimica | Video

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Una metodica di spettrometria di massa tandem (LC-MS / MS) cromatografica a liquido selettivo e sensibile, accoppiata con un&#39;estrazione efficiente di fase solida su una micropiastra a 96 pozzetti di MCX, è stata sviluppata per la misura della libera 3-nitrotirosina ( 3-NT) in urine umane con un elevato throughput, che è adatto per applicazioni cliniche.

Integrazione di estrazione miniaturizzata di fase solida e rilevazione LC-MS / MS di 3-Nitrotirosina nell&#39;urina umana per applicazioni cliniche

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

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9.2K Views.  Pharmasan Labs, Inc..  Una metodica di spettrometria di massa tandem (LC-MS / MS) cromatografica a liquido selettivo e sensibile, accoppiata con un'estrazione efficiente di fase solida su una micropiastra a 96 pozzetti di MCX, è stata sviluppata per la misura della libera 3-nitrotirosina ( 3-NT) in urine umane con un elevato throughput, che è adatto per applicazioni cliniche. 

Video: Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Preparazione del idrofobi Frameworks Metal-Organic tramite Plasma avanzata Chemical Vapor Deposition del perfluoroalcani per la rimozione di ammoniaca

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

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Chimica

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

Attivazione molecole, ioni e particelle solide con Acoustic Cavitazione

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Metodologia sintetico per Asymmetric Ferrocene derivati ​​sistemi bio-coniugati attraverso Solid Metodologia Resina a base di fase

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

Diffrazione di raggi X Polvere in Conservazione Scienza: verso Routine di cristallo Struttura Determinazione di corrosione prodotti sul patrimonio Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

Misura della dimensione delle particelle di distribuzione in torbidi soluzioni da Dynamic Light Scattering Microscopia

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Protocollo per la sintesi in fase solida di oligomeri di RNA contenente un 2&#39;-<em&gt; O</em&gt; -thiophenylmethyl Modifica e caratterizzazione attraverso il Dicroroo circolare

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

Un metodo pratico per estrazione ed analisi con cromatografia liquida dei neurotrasmettitori della catecolamina e loro metaboliti

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Surface Properties of Synthesized Nanoporous Carbon and Silica Matrices

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) with a 4.6 nm pore size, and ordered silica porous matrix, SBA-15, with a 5.3 nm pore size. This work describes the surface properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the differently ordered porous materials with similar pore sizes. For this purpose, OMC and SBA-15 with highly ordered nanoporous structures are synthesized via impregnation of the silica matrix by applying a carbon precursor and by the sol-gel method, respectively. The porous structure of investigated systems is characterized by an N2 adsorption-desorption analysis at 77 K. To determine the electrochemical character of the surface of synthesized materials, potentiometric titration measurements are conducted; the obtained results for OMC shows a significant pHpzc shift toward the higher values of pH, relative to SBA-15. This suggests that investigated OMC has surface properties related to oxygen-based functional groups. To describe the surface properties of the materials, the contact angles of liquids penetrating the studied porous beds are also determined. The capillary rise method has confirmed the increased wettability of the silica walls relative to the carbon walls and an influence of the pore roughness on the fluid/wall interactions, which is much more pronounced for silica than for carbon mesopores. We have also studied the melting behavior of D2O confined in OMC and SBA-15 by applying the dielectric method. The results show that the depression of the melting temperature of D2O in the pores of OMC is about 15 K higher relative to the depression of the melting temperature in SBA-15 pores with a comparable 5 nm size. This is caused by the influence of adsorbate/adsorbent interactions of the studied matrices.

Here we report the synthesis and characterization of ordered nanoporous carbon (with a 4.6 nm pore size) and SBA-15 (with a 5.3 nm pore size). The work describes the surface and textural properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the materials.

Proprietà della superficie di sintetizzato nanoporosi carbonio e matrici di silice

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

Sintesi in fase solida di [4,4] Spirocyclic ossime

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Development and Validation of Chromium Getters for Solid Oxide Fuel Cell Power Systems

Degradation of cathode in solid oxide fuel cells (SOFC) remains a major concern for the long-term performance stability and operational reliability. The presence of gas phase chromium species in air has demonstrated significant cathode performance degradation during long-term exposure due to unwanted compound formation at the cathode and electrolyte interface which retards the oxygen reduction reaction (ORR). We have demonstrated a novel method to mitigate the cathode degradation using chromium getters which capture the gas phase chromium species before it is ingested in the cathode chamber. Low-cost getter materials, synthesized from alkaline earth and transition metal oxides, are coated on the cordierite honeycomb substrate for application in the SOFC power systems. As-fabricated getters have been screened by chromium transpiration tests for 500 h in humidified air atmosphere in presence of chromium vapor. Selected getters have been further validated utilizing electrochemical tests. Typically, electrochemical performance of SOFCs (lanthanum strontium manganite (LSM) &#449; yttria stabilized zirconia (YSZ) &#449; Pt) was measured at 850 &#176;C in the presence and absence of Cr getter. For the 100 h cell tests containing getters, stable electrochemical performance was maintained, whereas the cell performance in the absence of Cr getters rapidly decreased in 10 h. Analyses of Nyquist plots indicated significant increase in the polarization resistance within the first 10 h of the cell operation. Characterization results from posttest SOFCs and getters have demonstrated the high efficiency of chromium capture for the mitigation of cell degradation.

Cathode poisoning from airborne contaminants in trace levels remains a major concern for long-term stability of high-temperature electrochemical systems. We provide a novel method to mitigate the cathode degradations using getters, which capture airborne contaminants at high temperature before entering electrochemically active stack area.

Sviluppo e convalida di Chromium Getters per sistemi di alimentazione a celle a combustibile a ossido solido

Degradation of cathode in solid oxide fuel cells (SOFC) remains a major concern for the long-term performance stability and operational reliability. The ...

Integrazione di estrazione miniaturizzata di fase solida e rilevazione LC-MS / MS di 3-Nitrotirosina nell'urina umana per applicazioni cliniche

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Capitoli in questo video

Preparazione del idrofobi Frameworks Metal-Organic tramite Plasma avanzata Chemical Vapor Deposition del perfluoroalcani per la rimozione di ammoniaca

Attivazione molecole, ioni e particelle solide con Acoustic Cavitazione

Metodologia sintetico per Asymmetric Ferrocene derivati sistemi bio-coniugati attraverso Solid Metodologia Resina a base di fase

Diffrazione di raggi X Polvere in Conservazione Scienza: verso Routine di cristallo Struttura Determinazione di corrosione prodotti sul patrimonio Art Objects

Misura della dimensione delle particelle di distribuzione in torbidi soluzioni da Dynamic Light Scattering Microscopia

Protocollo per la sintesi in fase solida di oligomeri di RNA contenente un 2'-

Un metodo pratico per estrazione ed analisi con cromatografia liquida dei neurotrasmettitori della catecolamina e loro metaboliti

Proprietà della superficie di sintetizzato nanoporosi carbonio e matrici di silice

Sintesi in fase solida di [4,4] Spirocyclic ossime

Sviluppo e convalida di Chromium Getters per sistemi di alimentazione a celle a combustibile a ossido solido

Integrazione di estrazione miniaturizzata di fase solida e rilevazione LC-MS / MS di 3-Nitrotirosina nell'urina umana per applicazioni cliniche

Riepilogo

Esplora Altri Video

Capitoli in questo video

Preparazione del idrofobi Frameworks Metal-Organic tramite Plasma avanzata Chemical Vapor Deposition del perfluoroalcani per la rimozione di ammoniaca

Attivazione molecole, ioni e particelle solide con Acoustic Cavitazione

Metodologia sintetico per Asymmetric Ferrocene derivati ​​sistemi bio-coniugati attraverso Solid Metodologia Resina a base di fase

Diffrazione di raggi X Polvere in Conservazione Scienza: verso Routine di cristallo Struttura Determinazione di corrosione prodotti sul patrimonio Art Objects

Misura della dimensione delle particelle di distribuzione in torbidi soluzioni da Dynamic Light Scattering Microscopia

Protocollo per la sintesi in fase solida di oligomeri di RNA contenente un 2'-

Un metodo pratico per estrazione ed analisi con cromatografia liquida dei neurotrasmettitori della catecolamina e loro metaboliti

Proprietà della superficie di sintetizzato nanoporosi carbonio e matrici di silice

Sintesi in fase solida di [4,4] Spirocyclic ossime

Sviluppo e convalida di Chromium Getters per sistemi di alimentazione a celle a combustibile a ossido solido

Metodologia sintetico per Asymmetric Ferrocene derivati sistemi bio-coniugati attraverso Solid Metodologia Resina a base di fase